Field of the Invention
The present invention relates to methods of generating highly concentrated proteins, e.g., highly concentrated antibodies or protein therapeutics. Specifically, the invention relates to methods of generating highly concentrated proteins comprising circulating a first protein solution through an ultrafiltration device while applying a flow of gas to the permeate side of a porous membrane within the ultrafiltration device, and collecting a second protein solution, wherein the second protein solution is highly concentrated, e.g., greater than about 260 grams of protein per liter of solution. The invention also relates to apparatus for use in the method of generating highly concentrated proteins.
Related Background Art
The step of concentrating a protein in a solution is often the final step in protein production and purification, and it is commonly a necessary step for both biotechnology and pharmaceutical applications. However, currently utilized methods of protein concentration have several significant disadvantages.
For example, some methods for concentrating solutions involve distillation processes, e.g., osmotic distillation (also known as osmotic evaporation) or thermal distillation. In thermal distillation, two chambers of the apparatus—one containing the feed solution (such as a solute-containing solution, e.g., a protein-containing solution) and another containing the distillate (such as a solute-free solvent, e.g., a protein-free solvent)—are separated by a porous hydrophobic membrane. See, e.g., Godino et al. (1996) J. Membr. Sci. 121:83-93; Khaeyet et al. (2000) J. Membr. Sci. 165:261-72. A temperature difference is maintained between the two chambers, such that the temperature of the feed-containing chamber is higher than the temperature of the distillate chamber. The temperature difference between the chambers causes a vapor pressure difference, which drives mass transfer from the feed-containing chamber to the distillate chamber. The gaseous phase is present only within the pores of the hydrophobic membrane. The major disadvantage of thermal distillation is that it is unsuitable for concentrating most protein solutions because high temperatures can damage protein solutes.
Osmotic distillation involves a similar principle to thermal distillation, except no temperature gradient is maintained between the two chambers. See, e.g., Kunz et al. (1996) J. Membr. Sci. 121:25-36. A hydrophobic porous membrane separates the two chambers, and the two chambers have different solute concentrations, which is the driving force for the mass transfer. The distillate chamber often contains high concentrations of solutes, e.g., salts, in order to maintain osmotic pressure differences. However, in this method, achievable protein concentrations are limited to the osmotic pressure of the salt solution. Additionally, the hydrophobic porous membrane creates an air-liquid interface, which can damage some proteins.
In a different method of protein concentration that does not employ osmotic or vapor pressure differences, a protein solution is pumped into an ultrafiltration device at high pressure, allowing solvent to flow through the membrane of the device while proteins are retained (see, e.g., FIG. 2). The protein-containing solution can be recirculated to a retentate tank as the solvent exits in the permeate. The solvent flow is caused by the difference in applied pressure between the retentate and the permeate sides of the device, typically about 10 to about 100 psig (pound-force per square inch gauge). When the concentration of the protein upstream of the membrane becomes high enough, the protein osmotic pressure becomes equal to the applied pressure gradient and the solvent flow stops (the gel-point). The disadvantage of this commonly used method is that this so-called “gel-point” limits the concentrations achievable with this method to a maximum of about 200 grams per liter for most proteins.
Thus, because of the limitations of existing protein concentration methods, there is a need for novel methods of producing highly concentrated protein solutions, e.g., a highly concentrated antibody solution, a highly concentrated therapeutic protein solution, etc. Protein concentrations are increased above the current range, e.g., about 50-200 g/L for an antibody solution, thus reducing storage volumes. In turn, freezer volume requirements are also reduced. The time and space required for freeze-drying (lyophilization) are reduced because there is up to, or greater than, approximately a 30% reduction in the volume of water that must be removed. A highly concentrated protein solution will increase the osmotic pressure of the solution, thereby preventing bacterial growth, and will increase viscosity of the protein solution, thereby increasing the residence time of the therapeutic protein in patients and increasing drug availability after administration.